Breast cancer is the second leading cause of cancer death in women in the United States. Invasion and metastasis are the most lethal characteristics of breast cancer and the leading cause of breast cancer-related death. TGF-beta normally inhibits breast cancer development by preventing mammary epithelial cell (MEC) proliferation, or by inducing MEC apoptosis. Mammary tumorigenesis negates the tumor suppressing activities of TGF-beta, thus facilitating TGF-beta to stimulate breast cancer invasion and metastasis. Fundamental gaps exist in our knowledge of how malignant MECs overcome the cytostatic actions of TGF-beta, and of how TGF-beta stimulates the development and progression of mammary tumors. These knowledge gaps have prevented science and medicine from developing treatments effective in antagonizing TGF-beta oncogenicity in developing and progressing breast cancers. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-beta type II receptor (TbetaR-II) antagonist that inhibits TGF-beta -stimulated gene expression, cell invasion, epithelial-to-mesenchymal transition, and morphological transformation in normal and cancer cells. Based on these and other preliminary findings, we hypothesize that delivering CystC (or CystC peptide mimetics) to developing breast cancers that are resistant to the cytostatic actions of TGF-beta will antagonize TGF-beta oncogenicity by (i) preventing TGF-beta binding to TbetaR-II; (ii) reducing TGF-beta: TbetaR-II binding to TaR-III and TbetaR-I; and (iii) selectively inhibiting Smad2/3-independent signaling pathways activated by TGF-beta. This hypothesis will be addressed by three Specific Aims. Specific Aim 1 will identify the CystC and TbetaR-II determinants that mediate CystC: TbetaR-II complex formation and mutants lacking these functions will be used to establish the role of CystC in selectively antagonizing TGF-beta isoform signaling in normal and malignant MECs. Specific Aim 2 will determine the TGF-beta signaling systems selectively inhibited by CystC, the mechanisms underlying these inhibitory reactions, and the impact of these reactions on TGF-beta function in normal and malignant MECs. Specific Aim 3 will determine whether CystC antagonizes TGF-beta oncogenicity and breast cancer progression in vivo by measuring the effects of CystC in preventing TGF-beta to stimulate breast cancer cell growth, invasion, angiogenesis, and metastasis, and to therapeutically combat these tumor behaviors in nude mice. These studies will provide valuable information on how TGF-beta promotes breast cancer invasion and metastasis, and, more importantly, on how to control these deadly processes by combating the oncogenicity of TGF-beta through the development and use of CystC-based TbetaR-II antagonists.